Application Of Zebrafish Model To Verify The Function Of SNP Locus Associated With Congenital Heart Disease

Congenital heart disease(CHD) is currently the most common birth defect worldwide, and has been the main cause of perinatal and infant mortality. Twenty-eight percent of all major congenital anomalies consist of heart defects. Epidemiologically, the worldwide prevalence of CHD is estimated to be 8 per 1,000 live births and 4 per 1,000 adults. In addition, patients with congenital heart disease usually requires long-term treatment, bringing heavy burden to their relatives and family on economic and emotion.During the past decade, studies of animal models have elucidated many fundamental pathways that genetically govern early cardiac patterns and differentiation. Epidemiological studies indicate that syndromic CHD comprises approximately 20% of cases. For example, the Holt-Oram syndrome, including congenital heart defects and limb abnormalities, can be found in the presence of gene mutations TBX5. The DiGeorge syndrome, including congenital heart disease and cleft palate, has a partial deletion of the gene TBX1.The remaining cases are represented by isolated non-syndromic CHM.In contrast to syndromic CHD, most non-syndromic CHD occurs sporadically and may result from a multifactorial inheritance model that involves a multitude of susceptibility genes with low-penetrance mutations or intermediate-penetrance mutations superposed onto unfavorable environmental factors.Even so, the case study of congenital heart disease in most sporadic is less and less, it is often overlooked some low penetrance susceptibility gene mutation. To identify common genetic variants associated with sporadic non-syndromic CHD in Han Chinese populations, a multistage genome-wide association study in CHD patients was performed, we found 53 SNPs(P<10-5). During the 500 kb region of these SNPs and where they located, we found 60 candidate genes, among which 32 genes were homologous one to one in zebrafish.In order to verify those susceptibility genes which related to the SNP sites to study their roles in the heart during the embryo development, we chose zebrafish as models and designed a kind of modified antisense oligonucleotides called morpholinos. We all designed morpholinos for 29 genes of the homologous genes.Morpholino often are used to knocking down gene function by connecting with mRNA or pre-mRNA. We microinjected the morpholinos into 1-4 cell stage zebrafish embryos, which their heart has green fluorescent in the fluorescence microscopy. The results showed that, the candidate susceptibility genes from GWAS in human congenital heart disease, such as grm4, iqgap2, tbx15, slc24a3,ptpn22,tbck, tbx3, their homologous genes deletion in zebrafish will lead to a variety of abnormal phenotypes in heart during zebrafish embryo development, such as atrial ventricular size changed, heart beat weakened, heart rate decreased, cyclization abnormal and tubular heart.A part of the candidate genes selected by GWAS have key roles in heart during zebrafish embryo development, providing a good research tool to further elucidate the mechanism of human CHD.